Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Determination of amino acids in saliva using capillary electrophoresis with fluorimetric detection

In the present study a sensitive method for the quantification of main free amino acids in saliva using capillary electrophoresis with laser induced fluorescence detection was developed. As background electrolyte 20 mM borate buffer pH 9.5 was used. Amino acids were separated after derivatization were optimized. The main amino acids occurring in saliva (Pro, Ser, Gly and Glu) were separated in less than 7 min. The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.1 to 2.4 nM. The developed method was employed for determination of amino acids in real saliva samples.     

Transvaginal hydrolaparoscopy for diagnosis of tubal infertility

OBJECTIVE: Infertility problem affects more than 70 million couples worldwide, 5-15% of which are couples in their reproductive age. Less and less invasive endoscopic methods like transvaginal hydrolaparoscopy have been developed by technological progress. This method enables not only precise identification, but is now increasingly used for treatment of tubal and peritoneal factor pathology, which cause approximately 35 per cent of female infertility. AIM: Evaluation of transvaginal hydrolaparoscopy (HLTV) usefulness for diagnosis of tubal infertility comparing to standard laparoscopy and hysterosalpingography (HSG). Results: In evaluation of patent fallopian tubes results of HLTV and HSG examinations are coincide in 87%, while obstruction diagnosed in HSG is confirmed only in 37% during HLTV examination. Transvaginal hydrolaparoscopy and HSG have similar sensitivity and specificity in diagnosis of hydrosalpinx, which is up to 100% . In comparison with HLTV histerosalpingography is less effective in evaluation of peritubal dilatations and adhesions. Both laparoscopic surgery and transvaginal laparoscopy have the same high sensitivity in diagnostics of the fallopian tubes patency and hydrosalpinx, which is up to 100%. In evaluation of peritubal adhesions and dilatations the results are very similar. Conclusions: 1. HLTV is a highly useful method in evaluation of the fallopian tubes pathologies which is significantly more sensitive than HSG in evaluation of such lesions as peritubal adhesions and obstructed fallopian tubes. 2. HLTV is as effective as laparoscopy in evaluation of patency and lesions of the fallopian tubes. 3. HLTV is a less invasive method, much better tolerated than laparoscopy and more suitable for the group of overweight patients. 4. Final assessment of HTLV technique will be possible following performance of a greater number of studies, where the foregoing conclusions present only initial observations.     

Fibroblast growth factor receptor signaling affects development and function of dopamine neurons - inhibition results in a schizophrenia-like syndrome in transgenic mice

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.     

Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.     

Apoptosis shifts to necrosis via intermediate types of cell death by a mechanism depending on c-myc and bcl-2 expression

Hypoxic and chemical hypoxia (antimycin A) commits cultured rat fibroblasts (Rat-1) towards apoptosis, necrosis or an intermediate form of cell death (aponecrosis) depending on the degree of hypoxia. Aponecrosis also occurs in vivo. Here, we demonstrate that c-myc and bcl-2, two proto-oncogenes known to lower or to enhance, respectively, the apoptotic threshold, also affect the type of cell death: apoptosis shifts to aponecrosis and aponecrosis to necrosis, depending on c-myc or bcl-2 expression and the antimycin A concentration (100–400 M). In cells with basal gene expression, apoptosis shifts to aponecrosis/necrosis at 300 M antimycin A (middle hypoxia). Overexpression of c-myc markedly increases cumulative cell death in response to antimycin A and lowers the antimycin A concentration required to shift apoptosis to aponecrosis/necrosis from 300 M to 100 M (low hypoxia). Overexpression of bcl-2 elicits the opposite effect, decreasing cumulative cell death in response to antimycin A and raising the drug concentration required to shift apoptosis to aponecrosis/necrosis to 400 M (high hypoxia). The passage from one to the other form of cell death involves various aponecrotic features with observed intermediate aspects between apoptosis and necrosis, a progressive increase in necrotic features being correlated with an increase in antimycin A concentration. The mechanism underlying the various effects of c-myc and bcl-2 on cell-death type has been related to the ability of these genes to counteract, to various extents, the ATP decrease occurring in response to different degrees of chemical hypoxia.The first two authors contributed equally to this workThis work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano), CNR/MIUR (Grant Progetto Finalizzato Oncologia), Ente Cassa di Risparmio di Firenze and Ministero dellIstruzione, dellUniversità e della Ricerca (MIUR, Cofin 2003). Andrea Lapucci is supported by a fellowship from the Federazione Italiana per la Ricerca sul Cancro (FIRC, Milano)     

The Non-Psychoactive Plant Cannabinoid,Cannabidiol Affects Cholesterol Metabolism-Related Genes in Microglial Cells

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that is clinically used in a 1:1 mixture with the psychoactive cannabinoid Δ⁹-tetrahydrocannabinol (THC) for the treatment of neuropathic pain and spasticity in multiple sclerosis. Our group previously reported that CBD exerts anti-inflammatory effects on microglial cells. In addition, we found that CBD treatment increases the accumulation of the endocannabinoid N-arachidonoyl ethanolamine (AEA), thus enhancing endocannabinoid signaling. Here we proceeded to investigate the effects of CBD on the modulation of lipid-related genes in microglial cells. Cell viability was tested using FACS analysis, AEA levels were measured using LC/MS/MS, gene array analysis was validated with real-time qPCR, and cytokine release was measured using ELISA. We report that CBD significantly upregulated the mRNAs of the enzymes sterol-O-acyl transferase (Soat2), which synthesizes cholesteryl esters, and of sterol 27-hydroxylase (Cyp27a1). In addition, CBD increased the mRNA of the lipid droplet-associated protein, perilipin2 (Plin2). Moreover, we found that pretreatment of the cells with the cholesterol chelating agent, methyl-β-cyclodextrin (MBCD), reversed the CBD-induced increase in Soat2 mRNA but not in Plin2 mRNA. Incubation with AEA increased the level of Plin2, but not of Soat2 mRNA. Furthermore, MBCD treatment did not affect the reduction by CBD of the LPS-induced release of the proinflammatory cytokine IL-1β. CBD treatment modulates cholesterol homeostasis in microglial cells, and pretreatment with MBCD reverses this effect without interfering with CBD’s anti-inflammatory effects. The effects of the CBD-induced increase in AEA accumulation on lipid-gene expression are discussed.     

The synthesis of acetyl-CoA by Clostridium thermoaceticum from carbon dioxide,hydrogen, coenzyme A and methyltetrahydrofolate

It has been demonstrated that enzymes from Clostridium thermoaceticum catalyze the following reaction in which Fd is ferredoxin and CH3THF is methyltetrahydrofolate. (for formula see text). The system involves hydrogenase, CO dehydrogenase, a methyltransferase, a corrinoid enzyme and other unknown components. Hydrogenase catalyzes the reduction of ferredoxin by H2; CO dehydrogenase then uses the reduced ferredoxin to reduce CO2 to a one-carbon intermediate that combines with CoASH and with a methyl group originating from CH3THF to form acetyl-CoA. It is proposed that these reactions are part of the mechanism which enables certain acetogenic autotrophic bacteria to grow on CO2 and H2.